Breast Cancer - High / Moderate Risk Sequencing Panel with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
|Test Code||Test Copy Genes||CPT Code Copy CPT Codes|
|BRCA1 and BRCA2||81162|
|Full Panel Price*||$540|
|Test Code||Test Copy Genes||Total Price||CPT Codes Copy CPT Codes|
|5433||Genes x (9)||$540||81162, 81321, 81323, 81404, 81405(x2), 81406(x2), 81408, 81479(x6)||Add|
We are happy to accommodate requests for testing single genes in this panel or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available.
This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 20 days.
The overall prevalence of germline BRCA1 or BRCA2 pathogenic variants in the general population is 1:400 to 1:800, with higher rates depending on the specific ethnicity, such as 1:40 in the Ashkenazi Jewish population. Nucleotide substitutions and small insertions/deletions are found in about 90% of individuals with an identifiable pathogenic variant. For individuals with pathogenic variants in these genes, BRCA1 pathogenic variants were observed in 63% and BRCA2 pathogenic variants in 37% (Petrucelli et al. 2016. PubMed ID: 20301425).
Pathogenic variants in genes other than BRCA1 and BRCA2 are often tested selectively, so that the proportion of breast and ovarian cancers with pathogenic variants in other genes is not known. Genes tested in this panel have been implicated in hereditary breast and ovarian cancer, and although individually these genes may be involved in a minority of inherited breast cancer genes, the combination of these high-risk genes may be responsible for a significant portion of these hereditary cancers (Turnbull and Rahman. 2008. PubMed ID: 18544032). A study by Walsh et al (2011) found approximately 6% of patients with hereditary ovarian cancers who do not have pathogenic variants in BRCA1 or BRCA2 have pathogenic variants in genes such as BRIP1, CHEK2, MRE11A, NBN, PALB2, RAD51C and TP53 (Walsh et al. 2011. PubMed ID: 22006311). Another study by Castéra et al. found that around a third of the deleterious variants they identified in their patient cohort were in genes outside of BRCA1/2, including CDH1, CHEK2, PALB2 and TP53 (Castéra et al. 2014. PubMed ID: 24549055).
Copy Number Variants (CNVs) are found in approximately 10% of individuals with an identifiable germline pathogenic variant, with 90% of these in BRCA1 and 10% in BRCA2 (Petrucelli et al. 2016. PubMed ID: 20301425). Clinical sensitivity for gross deletions/duplications for the other genes in Hereditary Breast and Ovarian Cancer is unknown. HBOC associated gross deletions have been reported for the PALB2 (Antoniou et al. 2014. PubMed ID: 25099575) and TP53 (Melhem-Bertrandt et al. 2012. PubMed ID: 21761402) genes. Approximately 1-2% of patients have large genomic deletions involving the ATM gene (Gatti. 2010. PubMed ID: 20301790).
Hereditary breast and ovarian cancer (HBOC) syndrome is an inherited disorder that is highly associated with tumors of the breasts and ovaries. Other malignancies in HBOC families can occur, including melanoma, pancreatic and prostate cancer. In comparison to sporadic breast and ovarian cancers, HBOC syndrome tends to occur at an earlier age (< 50 years), tumors often occur bilaterally, consist of multiple affected family members, including males with breast cancer, and occur with a higher predisposition in specific ethnicities, such as the Ashkenazi Jewish population (Petrucelli et al. 2016. PubMed ID: 20301425; Pruthi et al. 2010. PubMed ID: 21123638). Identifying individuals with a high risk for developing HBOC may allow for early detection of tumor formation and allow for prophylactic mastectomy and/or oophorectomy or other treatments (Smith. 2012. PubMed ID: 23050669). Breast and ovarian cancers can show familial inheritance due to shared environment or inherited genes of low penetrance, which confer a moderate risk (Berliner et al. 2013. PubMed ID: 23188549). In addition, approximately 5-10% of breast, and 10-15% of ovarian cancer cases are the result of genetic predisposition due to gene specific pathogenic variants that significantly increase an individual's risk of developing these cancers (Marchina et al. 2010. PubMed ID: 21042765). HBOC syndrome is mainly due to pathogenic variants in the BRCA1 and BRCA2 genes, however pathogenic variants have also been found in other genes. Higher incidences of breast and/or ovarian cancer have been observed in several syndromes, albeit with different cancer spectrums, including Li-Fraumeni syndrome, Cowden syndrome, Hereditary diffuse gastric cancer syndrome, Peutz-Jeghers syndrome, Ataxia-telangiectasia and Fanconi Anemia caused by pathogenic variants in TP53, PTEN, CDH1, STK11, ATM and PALB2, respectively.
Hereditary breast and ovarian cancer is inherited in an autosomal dominant manner and presents with high, although incomplete penetrance. Pathogenic variants in a number of genes have been reported to significantly increase an individual’s likelihood for developing breast cancer (Tan et al. 2008. PubMed ID: 18682420). Among those, germline pathogenic variants in the most common highly penetrant mutated breast cancer genes, BRCA1 and BRCA2 (Miki et al. 1994. PubMed ID: 7545954; Wooster et al. 1995. PubMed ID: 8524414), appear to provide the highest relative risk, ~10- to 20-fold. Hereditary BRCA1 and BRCA2 pathogenic variants account for 25-60% of inherited breast cancer (Pruthi et al. 2010. PubMed ID: 21123638; Meindl et al. 2011. PubMed ID: 21637635) and 11-39% of inherited ovarian cancer (Berliner et al. 2013. PubMed ID: 23188549). Large rearrangements (deletions, duplications, triplications), including the five most commonly reported BRCA1 CNVs (Hendrickson et al. 2005. PubMed ID: 15846789), can be detected using this test.
BRCA1 mutation carriers tend to have breast tumors that are estrogen receptor (ER) negative, progesterone receptor (PR) negative and basal type tumors, whereas BRCA2 mutation carriers have breast tumors that are ER positive, PR positive, and have a luminal phenotype (Pruthi et al. 2010. PubMed ID: 21123638). Individuals with HBOC with a more severe personal or family history tend to have pathogenic variants in BRCA1 vs. BRCA2 due to higher penetrance of pathogenic variants in the BRCA1 gene (Antoniou et al. 2000. PubMed ID: 10642429).
Other genes have also been implicated in hereditary breast and ovarian cancer, and although individual pathogenic variants in these genes may cause only a small fraction of inherited breast and ovarian cancer, the combination of moderately and mildly penetrant gene variants may be responsible for a significant portion of these hereditary cancers (Turnbull and Rahman. 2008. PubMed ID: 18544032). Genes related to other syndromes (Li-Fraumeni syndrome and Cowden syndrome) that are mutated and inherited in a dominant manner may predispose individuals to breast cancer and/or ovarian cancer with moderate penetrance. Early-onset breast cancer is a major component of Li-Fraumeni Syndrome (LFS), and pathogenic variants in the LFS-associated gene TP53 provide a 10- to 20-fold increased risk for developing bilateral mammary carcinomas, in addition to other cancers (including ovarian).
Individuals with Cowden syndrome caused by pathogenic variants in PTEN have a lifetime risk of 50% for breast cancer and 5-10% for endometrial cancer (Hearle et al. 2006. PubMed ID: 16707622; Eng. 2000. PubMed ID: 11073535).
CDH1 pathogenic variants predispose individuals to higher rates of breast cancer, but not ovarian cancer (Pennington and Swisher. 2012. PubMed ID: 22264603).
The relative risk of breast cancer due to PALB2 pathogenic variants has been estimated at 2.3, with a higher risk for women under 50 years of age (3.0 relative-risk) versus a lower risk in women older than 50 years of age (1.9 relative-risk) (Walsh and King. 2007. PubMed ID: 17292821). A larger study has found a higher relative risk (<9) depending on age for PALB2 mutation carriers, and the mean cumulative risk of breast cancer by 70 was 35% (Antoniou et al. 2014. PubMed ID: 25099575).
Female relatives of individuals with ataxia-telangiectasia and who are carriers of an ATM pathogenic variant have a 2-5 fold increased risk of breast cancer (Swift et al. 1987. PubMed ID: 3574400; Thompson et al. 2005. PubMed ID: 15928302). This is an important increase considering 0.2-1.0% of the general population are heterozygous for an ATM pathogenic variant (Lindor et al. 2008. PubMed ID: 18559331). See individual gene test descriptions for information on molecular biology of gene products and mutation spectra.
For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments.
For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.
This panel typically provides ≥98% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing.
Indications for Test
Individuals with a clinical presentation of Hereditary Breast and Ovarian Cancer are candidates. Clinical presentation includes family history, early-onset of breast cancer (< 50 years), bilateral breast tumors, multiple affected family members, including males with breast cancer, and a member of a high-risk ethnicity, such as the Ashkenazi Jewish population (Petrucelli et al. 2016. PubMed ID: 20301425; Pruthi et al. 2010. PubMed ID: 21123638). This is a predictive test which only provides information regarding the likelihood of breast and/or ovarian cancer. A positive test does not mean that a person will develop breast and/or ovarian cancer, and a negative test does not mean that a person will not. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - email@example.com
- Jerry Machado, PhD, DABMG, FCCMG - firstname.lastname@example.org
- Antoniou et al. 2000. PubMed ID: 10642429
- Antoniou et al. 2014. PubMed ID: 25099575
- Berliner et al. 2013. PubMed ID: 23188549
- Castéra et al. 2014. PubMed ID: 24549055
- Eng. 2000. PubMed ID: 11073535
- Gatti. 2010. PubMed ID: 20301790
- Hearle et al. 2006. PubMed ID: 16707622
- Hendrickson et al. 2005. PubMed ID: 15846789
- Lindor et al. 2008. PubMed ID: 18559331
- Marchina et al. 2010. PubMed ID: 21042765
- Meindl et al. 2011. PubMed ID: 21637635
- Melhem-Bertrandt et al. 2012. PubMed ID: 21761402
- Miki et al. 1994. PubMed ID: 7545954
- Pennington and Swisher. 2012. PubMed ID: 22264603
- Petrucelli et al. 2016. PubMed ID: 20301425
- Pruthi et al. 2010. PubMed ID: 21123638
- Smith. 2012. PubMed ID: 23050669
- Swift et al. 1987. PubMed ID: 3574400
- Tan et al. 2008. PubMed ID: 18682420
- Thompson et al. 2005. PubMed ID: 15928302
- Turnbull and Rahman. 2008. PubMed ID: 18544032
- Walsh and King. 2007. PubMed ID: 17292821
- Walsh et al. 2011. PubMed ID: 22006311
- Wooster et al. 1995. PubMed ID: 8524414
Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing.
For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).
(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign Variants
Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.
Deletion and Duplication Testing via NGS
As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.
In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.
Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.
When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).
We sequence all coding exons for each given transcript, plus ~10 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.
In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.
Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.
We cannot be certain that the reference sequences are correct.
Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.
We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.
myPrevent - Online Ordering
- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.